Project Summary Comprehensive mapping and monitoring of signaling pathways are essential for achieving the goals of precision medicine. Calcineurin (CN), the serine/threonine protein phosphatase and target of immunosuppressants, FK506 and CysA, is a critical mediator of Ca2+-dependent signaling with multiple functions relevant to human health. However, many CN-regulated substrates and processes remain to be elucidated. This proposal focuses on CNb1, a CN isoform with unique properties and functions that is conserved in vertebrates and broadly expressed in human tissues, but significantly under-studied. By addressing fundamental gaps in knowledge about CNb1, this research will discover novel CN-regulated signaling pathways, elucidate roles for CNb1 in healthy and diseased cells, and ultimately identify methods to therapeutically manipulate the enzyme. Our studies show that its unique C-tail, generated by alternative 3? end mRNA processing, confers distinct regulation and localization to CNb1: In vitro, maximal phosphatase activity of CNb1 in the presence of Ca2+/calmodulin is significantly lower than that of canonical CNb2, due to a unique C-terminal auto-inhibitory sequence that occludes an essential substrate binding site. In vivo, CNb1 localizes to membrane compartments, including the plasma membrane and Golgi, in contrast to canonical CN isoforms, which are primarily cytosolic. We show that lipidation of conserved cysteines in the CNAb1 C-tail promotes membrane association and that palmitoylation of CNAb1 is dynamic, suggesting a novel mechanism for regulating its distribution and activity in cells that will be examined in Aim 1. CNb1 does not dephosphorylate NFAT transcription factors and its substrates are currently unknown. Our central hypothesis is that by mapping CNb1-regulated signaling pathways we will uncover unique functions for CN at membranes and provide critical new insights into CN regulation in a broad range of tissues and processes. Our preliminary data, which suggests that CNb1 regulates synthesis of phosphatidylinositol 4-phosphate (PI4P) at the PM during GPCR signaling by dephosphorylating FAM126A, whose genetic disruption gives rise to hypomyelination and congenital cataracts (HCC), supports this hypothesis, which is further tested in Aim 2. We also propose innovative approaches, coupling TurboID for proximity labeling over fast time frames with computational identification of CN-binding peptides, to systematically discover additional CNb1 substrates and thus map this enzymes?s unique signaling network in Aim 3. We anticipate that this knowledge will have therapeutic applications for pathologies to which CN signaling contributes, and will create a critical new resource for researchers studying Ca2+- or phosphorylation-dependent signaling.